Several ways of making multimeric recombinant antibodies have been reported, which include the mini-antibody (Pack et al., Biotechnology 11:1271 (1993); Pack et al., Biochemistry 31:1579 (1992); Pack et al. J. Mol. Biol. 246:28 (1995); Rheinnecker et al., J. Immunol. 157:2989 (1996); and Pluckthun and Pack, Immunotechnology 3:83 (1997)), diabody-triabody-tetrabody (Hudson P J, Curr. Opin. Biotech. 9:395 (1998); Hiades et al., FEBS Lett. 409:437 (1997); Kortt et al., Protein Engineering 10:423 (1997); and Gall et al., FEBS Lett. 453:164 (1999)), protein A-fusion protein (Ito and Kurosawa, J. Biol. Chem. 268:20668 (1993)), streptavidin-fusion protein (Kipriyanov et al., Protein Engineering 9:203 (1996)), disulfide-linked fragments (Carter et al., Biotechnology 10:163 (1992)), fragments joined with chemically attached spacers (Cook and Wood, J. Immunol. Methods 171:227 (1994)), or post purification assembly of scFv multimers by denaturation and renaturation (Whitlow et al., Protein Engineering 7:1017 (1994) and U.S. Pat. No. 5,869,620).
Multimeric antibodies made by fusion with protein A or streptavidin may not be suitable for human use since protein A and streptavidin are highly immunogenic in humans. Linking antibody fragments by a disulfide bond can lead to a dimeric recombinant antibody. It has been reported that single chain antibody scFv-based molecules can sometimes be made in multimeric finals by changing the length of linker between the VH and VL domains in scFv. When the linker is longer than 3 but shorter than 12 amino acid residues, scFv can form dimers, called “diabodies;” when the linker is less than 2 residues, or when no linker residue is used, scFv can form either trimers or tetramers, called “triabodies” and “tetrabodies.” Since these multimeric scFv molecules are structurally constrained, little affinity improvement resulted from higher valency. Triabodies have identical or lower affinities than the diabodies, and tetrabodies have less than one fold higher affinity than the diabodies (Kortt et al., Protein Engineering 10:423 (1997); and Gall et al., FEBS Lett. 453:164 (1999)).